Mesenchymal stem cells (MSCs) are multipotent stem cells that have been isolated from numerous sources including human embryonic stem cells (hES). Derivation from hES is unique in that hES must be differentiated. In our hands, trypsinizing hES into single cells and plating them on gelatin coated plates in a DMEM medium supplemented with serum replacement media and FGF2 with either PDGF AB or EGF will induce differentiation of hES and selectively enhance the survival of MSCs over hES. Repeated passaging by trypsinization results in a highly enriched MSC culture. Enriched MSC cultures can be further purified to homogeneity by limiting dilution or FACS sorting for a CD105+ or CD73+ and CD24− cell population. The resulting hES-MSCs fulfill the ISCT minimal defining criteria for human MSCs, namely adherence to plastic, a surface antigen expression profile of CD29+, CD44+, CD49a+ CD49e+, CD73+, CD105+, CD166+, CD34−, CD45−, and a differentiation potential that includes adipogenesis, osteogenesis, and chondrogenesis. Finally, hES-MSCs can be extensively and stably propagated. This method of deriving hES-MSCs without the need for a xenogeneic feeder and use of animal serum could be used to derive clinically compliant MSCs from hESCs.
